Isolation and Derivation of Mouse Embryonic Germinal Cells

Summary

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Abstract

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos. Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF). Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days. The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state. Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

Protocol

Part 1: Pregnant Mouse Laparotomy Using cervical dislocation, euthanize a pregnant C57BL6J female mouse at 10.5-11.5 dpc. Clean the abdomen with antimicrobial soap, and then, shave it. After shaving, wash the abdomen with a saline solution. Then, dry the abdomen with a sterile gauze. Cover the mouse with a new sterile field. Make a ventral incision using forceps and dissection scissors. Identify and remove the entire uterus from the a…

Materials

Material Name	Type	Company	Catalogue Number	Comment
10.5 – 11.5 dpc pregnant C57BL6J female mouse crossed with the same background male mouse.
Mouse Fibroblast MEF-CF1 ATCC			SCRC-1040
Dulbecco’s Phosphate Buffered Saline (D-PBS)		Invitrogen	14190/086
Collagenase /Dispase Solution		Roche	269638
Gelatin		Sigma	G1890
Mitomycin C		Sigma	M4287
Trypsin EDTA		Invitrogen	25200-072
MEF Medium: Dulbecco Medium Eagle Modified (D-MEM) GLUTAMAX High glucose		Invitrogen	10566024
10% Fetal Bovine Serum		Invitrogen	16000/044
1% of antibiotic-antimycotic		Invitrogen	15420/096
Knockout Media: 80% knockout D-MEM		Invitrogen	10829/018
20% Knockout serum Invitrogen Cat. No. 1028/028, 200mM L-glutamine Invitrogen			25130/081	with β-mercaptoethanol Sigma Catalogue Number: M7522
1X non essential amino acid solution		Invitrogen	11140/050
1% antibiotic-antimycotic		Invitrogen	15420/096	supplemented with grow factor as 2500 U of Leukemia inhibitory factor (mLIF-ESGRO) Millipore Catalogue Number: ESG1106
40 ng/ml of Recombinant Murine Stem Cell Factor (SCF) ReproTech			250-03
20 ng/ml of Basic Fibroblast Growth factor (bFGF)		Invitrogen	13256-029
Corning center well culture dish 60mm		Fisher	07-200-79	1.5 ml eppendorf tube, and 15 ml falcon tubes

Forceps No. 14 and 15, scalpel 35 mm, dissection scissors, surgical fields, antimicrobial soap, saline solution 0.9 %, gauze, ice foam container, razor blade, scalpel 35 mm, fine forceps No. 4 and 5, fine teasing needle with handle, filter paper sheets, and pulled glass pipettes.

Equipment: dissecting stereomicroscope, light source Schott Fostec, inverted microscope, micropipette 10 and 200ul, centrifuge, bio safety Cabinet.